Figure 5. Suppression of IL-27 expression in DC contributes to Opn-i-mediated Th17 differentiation.

(A) BM-derived DC (Ifnar1^+/+ [●] vs. Ifnar1^−/− [○]) were incubated (2×10⁶ DC/ml) with the indicated concentration of LPS before 30h supernatants were evaluated for IL-27p28 amounts by ELISA in triplicate well; representative of 3 experiments. (B) BM-derived DC (Opn wt [●] vs. Opn^−/− [○]) incubated with rIFNα (5×10⁶ DC/ml). 42h later IL-27-p28 amounts were determined and represent 2 experiments. (C) BM-derived DC incubated ×48h before IL-27-p28 amounts were determined by ELISA in triplicate wells. (left panel) Comparison of IL-27 secretion by Ifnar1^+/+ and Ifnar1^−/− DC (10⁷ DC/ml) in 44h culture supernatants. (middle panel) Comparison of IL-27 secretion by Opn wt and Opn-deficient (Spp1^−/−) DC (2×10⁶ DC/ml). (right panel) Comparison of IL-27 secretion by Opn-deficient DC transduced with lenti-GFP (control) or lenti-ΔOpn-i (2×10⁶ DC/ml) in 24h culture supernatants; data representative of 2 experiments. (D) Comparison of the amounts of IL-27p28 mRNA in resting BM-derived DC from Ifnar1^+/+ or Ifnar1^−/− donors. Real-time PCR of total cDNA and results obtained from cycling threshold (−ΔΔCt) values normalized to β-actin. (E–F) Two different Th17 skewing conditions were tested: (upper panel) rIL-23 and IFNγ + IL-4 Abs; (lower panel) rIL-6 and rTGFβ. (E) 2D2 T cells were incubated with Opn-deficient BM-DC, 1 μg/ml MOG peptide and 20 μg/ml IL-27 Ab or isotype IgG. 24h after T cell re-stimulation with soluble CD3 Ab, the amount of IL-17 was measured (mean ± SEM) and is representative of 4 experiments. (F) DC and OT-2 T cell co-cultures under the same skewing conditions used in (E) were treated with rIFNα with or without IL-27 Ab (20 μg/ml) and stimulated with OVA peptide ×5d, followed by 24h T cell re-stimulation with soluble CD3 Ab. The amount of IL-17 analyzed by ELISA in triplicate wells is shown (mean ± SEM); data are representative of 2 experiments.